Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.368
Filtrar
1.
An Bras Dermatol ; 94(4): 429-433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31644615

RESUMO

BACKGROUND: Behçet disease is a prototypical systemic autoimmune disease, caused by a complex interplay between environmental and genetic factors. The transmembrane immunoglobulin and mucin domain-3 (TIM-3) is a distinct member of the TIM family that is preferentially expressed on Th1 cells and plays a role in Th1-mediated autoimmune or inflammatory diseases, such as Behçet disease. OBJECTIVE: The aim of this study was to test the potential association between TIM-3 gene polymorphisms and Behçet disease. METHODS: Two single-nucleotide polymorphisms of TIM-3 (rs9313439 and rs10515746) were genotyped in 212 patients with Behçet disease and 200 healthy controls. Typing of the polymorphisms was performed using multiplex PCR amplification. RESULTS: There were no significant differences in allele and genotype frequencies between the Behçet disease patients and controls who were successfully genotyped. Similar results were also found after stratification by gender, age, or clinical features. STUDY LIMITATIONS: Lack of studies on various racial or ethnic groups and small sample size. CONCLUSION: This study failed to demonstrate any association between the tested TIM-3 polymorphisms and Behçet disease.


Assuntos
Síndrome de Behçet/genética , Receptor Celular 2 do Vírus da Hepatite A/genética , Polimorfismo de Nucleotídeo Único , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Frequência do Gene , Estudos de Associação Genética , Humanos , Irã (Geográfico) , Modelos Logísticos , Masculino , Reação em Cadeia da Polimerase Multiplex , Medição de Risco , Fatores de Risco
2.
J Med Microbiol ; 68(11): 1641-1648, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31526456

RESUMO

Introduction: Onychomycosis is a debilitating, difficult-to-treat nail fungal infection with increasing prevalence worldwide. The main etiological agents are dermatophytes, which are common causative pathogens in superficial fungal mycoses. Conventional detection methods such as fungal culture have low sensitivity and specificity and are time-consuming.Aim: The main objective of this study was to design, develop and validate a real-time probe-based multiplex qPCR assay for the detection of dermatophytes and Fusarium species.Methodology: The performance characteristics of the qPCR assays were evaluated. The multiplex qPCR assays targeted four genes (assay 1: pan-dermatophytes/Fusarium spp.; assay 2: Trichophyton rubrum/Microsporum spp.). Analytical validation was accomplished using 150 fungal isolates and clinical validation was done on 204 nail specimens. The performance parameters were compared against the gold standard (fungal culture) and expanded gold standard (culture in conjunction with sequencing).Results: Both the single-plex and multiplex qPCR assays performed well especially when compared against the expanded gold standard. Among the 204 tested nail specimens, the culture method showed that 125 (61.3 %) were infected with at least one organism, of which 40 yielded positive results for dermatophytes and Fusarium spp. These target organisms detected include 20 dermatophytes and 22 Fusarium spp. The developed qPCR assays demonstrated excellent limit of detection, efficiency, coefficient of determination, analytical and clinical sensitivity and specificity.Conclusion: The multiplex qPCR assays were reliable for the diagnosis of onychomycosis, with shorter turn-around time as compared to culture method. This aids in the planning of treatment strategies to achieve optimal therapeutic outcome.


Assuntos
Arthrodermataceae/isolamento & purificação , Dermatomicoses/microbiologia , Fusariose/microbiologia , Fusarium/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Arthrodermataceae/classificação , Arthrodermataceae/genética , Dermatomicoses/diagnóstico , Fusariose/diagnóstico , Fusarium/genética , Humanos , Sensibilidade e Especificidade
3.
BMC Infect Dis ; 19(1): 778, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488066

RESUMO

BACKGROUND: A diagnostic method to simultaneously detect and discriminate porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3) in clinical specimens is imperative for the differential diagnosis and monitoring and control of PCVs in the field. METHODS: Three primer pairs were designed and used to develop a multiplex PCR assay. And 286 samples from 8 farms in Hubei province were tested by the developed multiplex PCR assay to demonstrate the accuracy. RESULTS: Each of target genes of PCV1, PCV2 and PCV3 was amplified using the designed primers, while no other porcine viruses genes were detected. The limit of detection of the assay was 10 copies/µL of PCV1, PCV2 OR PCV3. The results of the tissue samples detection showed that PCV1, PCV2 and PCV3 are co-circulating in central China. The PCV1, PCV2 and PCV3 singular infection rate was 52.4% (150/286), 61.2% (175/286) and 45.1% (129/286), respectively, while the PCV1 and PCV2 co-infection rate was 11.2% (32/286), the PCV1 and PCV3 co-infection rate was 5.9% (17/286), the PCV2 and PCV3 co-infection rate was 23.4% (67/286), and the PCV1, PCV2 and PCV3 co-infection rate was 1.7% (5/286), respectively, which were 100% consistent with the sequencing method and real-time PCR methods. CONCLUSIONS: The multiplex PCR assay could be used as a differential diagnostic tool for monitoring and control of PCVs in the field. The results also indicate that the PCVs infection and their co-infection are severe in Hubei province, Central China.


Assuntos
Infecções por Circoviridae/diagnóstico , Circovirus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Animais , China , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/virologia , Circovirus/classificação , Circovirus/genética , Diagnóstico Diferencial , Genes Virais , Incidência , Tipagem Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/epidemiologia , Virologia/métodos
4.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 904-910, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31484252

RESUMO

Objective: To analyze the etiologic and epidemiological characteristics of adult acute respiratory infections in Shanghai during 2015-2017. Methods: Data was collected from outpatients with acute respiratory infections who visited the Fever Clinics in three hospitals of different levels in three administrative regions of Shanghai, from 2015 to 2017. Basic information and nasopharyngeal swabs were collected from cases in line with the inclusion criteria. Multiplex RT-PCR and bacterial cultures were performed to detect the respiratory pathogens. Results: A total of 806 individuals were enrolled from 2015 to 2017. Respiratory pathogens were identified in 73.45% (592/806) of the cases, with the virus detection rate as 66.75% (538/806). It was found that the major respiratory pathogens for virus detection were influenza A in 326 (40.45%), influenza B in 116 (14.39%), rhinovirus/enterovirus in 39 (4.84%) of the cases. The overall detection rate of bacteria was 16.13% (130/806), including Klebsiella pneumoniae in 90 (11.17%) cases, Staphylococcus Aureus in 46 (5.71%) cases. Other kind of bacteria were not detected in our study. The detection rates on Mycoplasma pneumoniae was 5.33% (43/806) and on Chlamydia pneumonia was 0.37% (3/806). Co-infection with multiple pathogens was detected in 18.61% (150/806) of the cases, including 135 with double infection (accounting for 90.00%), 14 with triple infection and 1 with quadruple infection (accounted for 9.33% and 0.67%, respectively). Among the 150 cases with co-infections, the main identified pathogens were influenza A, Klebsiella pneumoniae, Staphylococcus aureus, and Mycoplasma pneumoniae. Pathogens of acute respiratory infections that identified among the outpatients from the Fever Clinics at different time, region or population, the characteristics were different (P<0.001). Conclusions: In 2015-2017, outpatients with acute respiratory infections in Shanghai were mainly caused by influenza virus or other viruses, however dynamically with its composition, time, region and characteristics of the population. It is necessary to strengthen and combine related medical and preventive services and to develop the appropriate strategies regarding clinical diagnosis and treatment.


Assuntos
Bactérias/isolamento & purificação , Infecções Bacterianas/diagnóstico , Influenza Humana/diagnóstico , Reação em Cadeia da Polimerase Multiplex/métodos , Nasofaringe , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/etiologia , Viroses/diagnóstico , Vírus/isolamento & purificação , Doença Aguda , Adulto , Bactérias/genética , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/microbiologia , China/epidemiologia , Coinfecção/diagnóstico , Enterovirus/genética , Enterovirus/isolamento & purificação , Monitoramento Epidemiológico , Humanos , Incidência , Vírus da Influenza A/genética , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/epidemiologia , Influenza Humana/virologia , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Mycoplasma pneumoniae , Nasofaringe/microbiologia , Nasofaringe/virologia , Vigilância da População , Infecções Respiratórias/diagnóstico , Rhinovirus/genética , Rhinovirus/isolamento & purificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Viroses/epidemiologia , Viroses/virologia , Vírus/genética
5.
Arch Virol ; 164(11): 2761-2768, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31506786

RESUMO

A multiplex polymerase chain reaction (mPCR) assay was developed to detect and distinguish feline panleukopenia virus (FPV), feline bocavirus (FBoV) and feline astrovirus (FeAstV). Three pairs of primers were designed based on conserved regions in the genomic sequences of the three viruses and were used to specifically amplify targeted fragments of 237 bp from the VP2 gene of FPV, 465 bp from the NP1 gene of FBoV and 645 bp from the RdRp gene of FeAstV. The results showed that this mPCR assay was effective, because it could detect at least 2.25-4.04 × 104 copies of genomic DNA of the three viruses per µl, was highly specific, and had a good broad-spectrum ability to detect different genotypes of the targeted viruses. A total of 197 faecal samples that had been screened previously for FeAstV and FBoV were collected from domestic cats in northeast China and were tested for the three viruses using the newly developed mPCR assay. The total positive rate for these three viruses was 59.89% (118/197). From these samples, DNA from FPV, FBoV and FeAstV was detected in 73, 51 and 46 faecal samples, respectively. The mPCR testing results agreed with the routine PCR results with a coincidence rate of 100%. The results of this study show that this mPCR assay can simultaneously detect and differentiate FPV, FBoV and FeAstV and can be used as an easy, specific and efficient detection tool for clinical diagnosis and epidemiological investigation of these three viruses.


Assuntos
Bocavirus/genética , Proteínas do Capsídeo/genética , Vírus da Panleucopenia Felina/genética , Mamastrovirus/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , Bocavirus/isolamento & purificação , Doenças do Gato/diagnóstico , Doenças do Gato/virologia , Gatos , China , Primers do DNA/genética , Fezes/virologia , Vírus da Panleucopenia Felina/isolamento & purificação , Mamastrovirus/isolamento & purificação , Filogenia , Análise de Sequência de DNA
6.
BMC Infect Dis ; 19(1): 827, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547805

RESUMO

BACKGROUND: The worldwide expansion of macrolide-resistant Mycoplasma genitalium (MG) in cases of genital infections has led to an increased recurrence rate of these infections after first-line azithromycin treatment. By detecting the presence of azithromycin-resistant MG, the patient's antibiotic treatment can be targeted and the spread of resistance prevented. With this aim in mind, macrolide-resistance detection kits are helpful tools for the physician. METHODS: Azithromycin resistance mutations in MG are targeted using a four-color multiplex real-time RT-PCR assay. Tested targets include plasmid DNA (as positive controls) as well as macrolide-sensitive and macrolide-resistant genomic DNA from characterized cell lines and clinical samples. RESULTS: The analytical data presented here were generated from plasmid DNA and genomic RNA/DNA and include adaptation to an internal control, specificity between targets, specificity vs non-MG species, limit of detection (LoD) and interference studies (co-infection and endogenous substances). The clinical data were based on the application of the assay to clinical samples characterized by sequencing. CONCLUSIONS: A new NAAT (nucleic acid amplification test) prototype has been developed in collaboration with the Diagenode s.a. company, this prototype targets MG and azithromycin-resistance mutations in that pathogen.


Assuntos
Azitromicina/farmacologia , Farmacorresistência Bacteriana/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Mycoplasma genitalium/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Antibacterianos/farmacologia , Humanos , Infecções por Mycoplasma/tratamento farmacológico , Infecções por Mycoplasma/microbiologia , Especificidade por Substrato
7.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 17(2): 71-76, ago. 2019. tab, ilus
Artigo em Espanhol | LILACS, BDNPAR | ID: biblio-1008486

RESUMO

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)


STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)


Assuntos
Animais , Toxina Shiga , Escherichia coli , Reação em Cadeia da Polimerase Multiplex
8.
Infect Dis Poverty ; 8(1): 68, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31362789

RESUMO

BACKGROUND: Echinococcosis caused by Echinococcus is one of the most major infectious diseases in north-west highland of China. E. granulosus sensu strict, E. multilocularis, and E. canadensis are known to be the only three species related to human health transmitting in the areas. To achieve targeted treatment and control of echinococcosis, the accurate identification and discrimination of the species are important. However, currently the available diagnostic approaches do not present ideal results either in accuracy or efficiency. METHODS: In the study, a set of primers were designed to aim at the three human-pathogenic Echinococcus species in China. The one-step multiplex PCR assay was developed and evaluated for the specificity and sensitivity. A total of 73 parasitic lesions and 41 fecal materials obtained from human and various animals collected in the clinic and the field were tested to assess the applicability of this method. RESULTS: The multiplex PCR effectively detected the individual DNA from the targeted species and their random mixtures generating with distinguishable expected size of products. The detection limit of the assay for each of the three species was 5 pg/µl when they were tested separately. When DNA mixtures of the targeted species containing the same concentration were used as templates, the lowest amount of DNA which can be detected was 50 pg/µl, 10 pg/µl and 5 pg/µl for E. granulosus s. s., E. multilocularis, and E. canadensis respectively. No cross-reactivity was observed when DNA from eight genetically close species was used as control templates. The multiplex PCR identifications of all samples were in line with the original sequencing results except for those infected with E. shiquicus, which showed negative signals in the developed assay. Of all the tested stool materials, 16 were previously found positive for Echinococcus by visual and microscopic examination. Among these 16 samples, 13 were confirmed by the multiplex PCR, and the other three tested negative. Additionally, the multiplex PCR identified another 14 positive feces from the remained 25 stool samples which absence of worms. CONCLUSIONS: The developed multiplex PCR shows advantages in fast diagnosis and large-scale epidemiological investigation, which proven to be a promising tool utilized in clinic and surveillance system.


Assuntos
Equinococose/diagnóstico , Echinococcus/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Animais , China , Diagnóstico Diferencial , Equinococose/classificação , Equinococose/parasitologia , Echinococcus granulosus/isolamento & purificação , Echinococcus multilocularis/isolamento & purificação , Humanos
9.
Microbiol Res ; 227: 126298, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421716

RESUMO

An increasing number of infections originating from probiotic use are reported worldwide, with the majority of such cases caused by the yeast Saccharomyces 'boulardii', a subtype of S. cerevisiae. Reliably linking infectious cases to probiotic products requires unequivocal genotyping data, however, these techniques are often time-consuming and difficult to implement in routine diagnostics. This leads to a widespread lack of genetic data regarding the origin of Saccharomyces infections. We propose a quick and reliable PCR-based protocol for the identification of S. 'boulardii' based on a combined analysis of interdelta fingerprinting and microsatellite typing. By applying various typing methods and our proposed method to the clinical yeast collection of a Hungarian hospital we show that probiotic origin is common among clinical Saccharomyces, and that the new multiplex method enables rapid and unequivocal identification of probiotic yeast infections. This method can be applied for the identification of yeast infection sources, helping decisions on probiotic use.


Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Técnicas de Tipagem Micológica/métodos , Probióticos , Saccharomyces/genética , Saccharomyces/isolamento & purificação , DNA Fúngico/isolamento & purificação , Fungemia/microbiologia , Técnicas de Genotipagem , Humanos , Repetições de Microssatélites , Micoses/microbiologia , Saccharomyces/classificação , Saccharomyces/patogenicidade , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/isolamento & purificação
10.
BMC Med Genet ; 20(1): 139, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31412794

RESUMO

BACKGROUND: Duchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and "next-generation" sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients' MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations. METHODS: In this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations. RESULTS: DMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis. CONCLUSION: Our results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Testes Genéticos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Pré-Escolar , Éxons , Feminino , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Mutação
11.
Rev Inst Med Trop Sao Paulo ; 61: e37, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31411267

RESUMO

Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.


Assuntos
Candida glabrata/genética , Candidíase/microbiologia , DNA Fúngico/genética , Técnicas de Tipagem Micológica/métodos , Candida glabrata/isolamento & purificação , DNA Ribossômico/genética , DNA Espaçador Ribossômico/genética , Feminino , Humanos , Masculino , México , Reação em Cadeia da Polimerase Multiplex , Filogenia , Análise de Sequência de DNA
12.
BMC Infect Dis ; 19(1): 630, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31315581

RESUMO

BACKGROUND: In hospitalised patients with diarrhoea a positive campylobacter stool Polymerase Chain Reaction (PCR) test with negative culture results as well as Enteropathogenic Escherichia coli (EPEC) positive stool PCRs, challenges the clinician and may lead the unexperienced clinician astray. The aim of the study was to elucidate the clinical significance of positive Campylobacter and/or EPEC test results in hospitalised patients with diarrhoea. METHODS: We conducted a retrospective case-case study. Case groups with 1) EPEC only and 2) EPEC in combination with any other pathogen in the PCR multiplex array, 3) PCR positive/culture negative Campylobacter, and 4) PCR positive/culture positive Campylobacter were compared. Medical records were reviewed and cases classified according to pre-specified clinical criteria as infectious gastroenteritis or non-infectious causes for diarrhoea. We analyzed the association between laboratory findings (the 4 subgroups) and the pre-specified clinical classification. We further sequenced culture negative campylobacter samples and tested EPEC for bundle forming pilus A (bfpA) gene, distinguishing typical from atypical EPEC. RESULTS: A total of 291 patients were included, 169 were PCR positive for Campylobacter and 122 for EPEC. For both pathogens, co-infections were more common in culture negative/PCR positive samples than in culture positive samples. Clinical characteristics differed significantly in and between groups. Campylobacter culture positive patients had very high prevalence of characteristics of acute infectious gastroenteritis, whereas patients with PCR positive test results only often had an alternative explanation for their diarrhoea. Culture positives were almost exclusively C. jejuni/coli, whereas in culture negatives, constituting a third of the total PCR positives, C. concisus was the most frequent species. The vast majority of EPEC only positives had documented non-infectious factors that could explain diarrhoea. The EPEC co-infected group mimicked the culture positive campylobacter group, with most patients fulfilling the infectious gastroenteritis criteria. CONCLUSIONS: In hospitalised patients, positive PCR results for campylobacter and EPEC should be interpreted in a clinical context after evaluation of non-infectious diarrhoea associated conditions, and cannot be used as a stand-alone diagnostic tool.


Assuntos
Escherichia coli Enteropatogênica/genética , Infecções por Escherichia coli/microbiologia , Fezes/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Idoso , Técnicas Bacteriológicas , Campylobacter/genética , Campylobacter/patogenicidade , Diarreia/microbiologia , Escherichia coli Enteropatogênica/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Feminino , Gastroenterite/epidemiologia , Gastroenterite/microbiologia , Hospitalização/estatística & dados numéricos , Humanos , Masculino , Pessoa de Meia-Idade , Noruega/epidemiologia , Estudos Retrospectivos
13.
BMC Infect Dis ; 19(1): 571, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266450

RESUMO

BACKGROUND: Carbapenemase-producing organisms (CPOs) have emerged as antibiotic-resistant bacteria of global concern. Here we assessed the performance of the Carba (beta) assay, a multiplex real-time PCR assay developed by SpeeDx for the detection of key carbapenemase-encoding genes: KPC, NDM, OXA-48-like, IMP-4-like, and VIM. METHODS: DNA extracts of 180 isolates were tested with the Carba (beta) assay, using previously validated in-house TaqMan probe assays for the relevant carbapenemase genes as the reference standard. The Carba (beta) assay was then directly used to screen 460 DNA extracts of faecal specimens, with positive results subjected to the aforementioned in-house assays plus Sanger sequencing. RESULTS: The Carba (beta) assay correctly identified the presence of the respective carbapenemase genes in 154 of 156 isolates and provided negative results for all 24 non-CPO isolates. Two isolates provided positive results for OXA-48-like carbapenemase by the Carba (beta) assay only. The Carba (beta) assay had sensitivities of 100% for all targets, and specificities of 100% for KPC, NDM, IMP-4-like, and VIM targets, and 98.5% for OXA-48-like targets. When applied directly to faecal specimens, eight samples were positive by the Carba (beta) assay, two of which were confirmed by in-house TaqMan probe PCR or DNA sequencing. CONCLUSIONS: The Carba (beta) assay is highly sensitive and specific for detecting key carbapenemase genes in isolates. Further testing is required to assess this assay's suitability for direct screening of clinical specimens.


Assuntos
Bactérias/genética , Proteínas de Bactérias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , beta-Lactamases/genética , Antibacterianos , Bactérias/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Humanos , Sensibilidade e Especificidade
14.
J Clin Pathol ; 72(12): 810-816, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31300531

RESUMO

AIMS: Leiomyosarcomas (LMSs) occur in various tissues and harbour potential for metastases. The genomic landscape of LMS is poorly understood. In an effort to improve understanding of the LMS genome, we analysed 11 LMSs of somatic soft tissue including matching tissue of normal phenotype. METHODS: DNA derived from microdissected tumour domains and matching normal tissue underwent amplicon sequencing of 409 tumour suppressors and oncogenes using the Ion Torrent Comprehensive Cancer Panel. RESULTS: Genomic changes were heterogeneous with few recurrent abnormalities detected. Coding variants were identified in genes involved in signal transduction, transcriptional regulation and DNA repair. There were variants in several genes related to angiogenesis and GPR124 variants (TEM5) were confirmed by immunohistochemical analysis of a LMS tissue microarray. Surprisingly, there were shared coding variants in tumour and corresponding normal tissue. CONCLUSIONS: LMSs are a very heterogeneous population lacking recurrent somatic abnormalities. The presence of damaging mutations in normal tissue may reflect either a germline predisposition or field effect rather than tissue contamination. Hopeful therapeutic targets appear to be those related to AKT/MTOR pathway.


Assuntos
Biomarcadores Tumorais/genética , Análise Mutacional de DNA/métodos , Dosagem de Genes , Leiomiossarcoma/genética , Reação em Cadeia da Polimerase Multiplex , Mutação , Neoplasias de Tecidos Moles/genética , Biomarcadores Tumorais/análise , Variações do Número de Cópias de DNA , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Leiomiossarcoma/química , Leiomiossarcoma/patologia , Leiomiossarcoma/terapia , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Neoplasias de Tecidos Moles/química , Neoplasias de Tecidos Moles/patologia , Neoplasias de Tecidos Moles/terapia
15.
BMC Vet Res ; 15(1): 253, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324180

RESUMO

BACKGROUND: Avian influenza virus (AIV), infectious bronchitis virus (IBV), and Newcastle disease virus (NDV) are important avian pathogens that can cause enormous economic loss on the poultry industry. Different respiratory etiological agents may induce similar clinical signs that make differential diagnosis difficult. Importantly, AIV brings about severe threat to human public health. Therefore, a novel method that can distinguish these viruses quickly and simultaneously is urgently needed. RESULTS: In this study, an oligonucleotide microarray system was developed. AIV, including H5, H7, and H9 subtypes; NDV; and IBV were simultaneously detected and differentiated on a microarray. Three probes specific for AIV, NDV, and IBV, as well as three other probes for differentiating H5, H7, and H9 of AIV, were first designed and jet-printed to predetermined locations of initiator-integrated poly(dimethylsiloxane) for the synchronous detection of the six pathogens. The marked multiplex reverse transcription polymerase chain reaction (PCR) products were hybridized with the specific probes, and the results of hybridization were read directly with the naked eyes. No cross-reaction was observed with 10 other subtypes of AIV and infectious bursal disease virus, indicating that the oligonucleotide microarray assay was highly specific. The sensitivity of the method was at least 100 times higher than that of the conventional PCR, and the detection limit of NDV, AIV, H5, H7, and H9 can reach 0.1 EID50 (50% egg infective dose), except that of IBV, which was 1 EID50 per reaction. In the validation of 93 field samples, AIV, IBV, and NDV were detected in 53 (56.99%) samples by oligonucleotide microarray and virus isolation and in 50 (53.76%) samples by conventional PCR. CONCLUSIONS: We have successfully developed an approach to differentiate AIV, NDV, IBV, H5, H7, and H9 subtypes of AIV using oligonucleotide microarray. The microarray is an accurate, high-throughput, and relatively simple method for the rapid detection of avian respiratory viral diseases. It can be used for the epidemiological surveillance and diagnosis of AIV, IBV, and NDV.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Influenza A/genética , Vírus da Doença de Newcastle/genética , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Doenças das Aves Domésticas/virologia , Animais , Infecções por Coronavirus/virologia , Vírus da Doença Infecciosa da Bursa/genética , Influenza Aviária/virologia , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Multiplex/veterinária , Doença de Newcastle/virologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Aves Domésticas , Sensibilidade e Especificidade
16.
BMC Infect Dis ; 19(1): 618, 2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299893

RESUMO

BACKGROUND: The increased transmission of multidrug-resistant (MDR) tuberculosis (TB) poses a challenge to tuberculosis prevention and control in Sri Lanka. Isoniazid (INH) is a key element of the first line anti tuberculosis treatment regimen. Resistance to INH may lead to development of MDR TB. Therefore, early detection of INH resistance is important to curb spread of resistance. Due to the limited availability of rapid molecular methods for detection of drug resistance in Sri Lanka, this study was aimed at developing a simple and rapid gold nanoparticle (AuNP) based lateral flow strip for the simultaneous detection of the most common INH resistance mutation (katG S315 T, 78.6%) and Mycobacterium tuberculosis (MTb). METHODS: Lateral flow strip was designed on an inert plastic backing layer containing a sample pad, nitrocellulose membrane and an absorption pad. Biotin labeled 4 capture probes which separately conjugated with streptavidin were immobilized on the nitrocellulose. The test sample was prepared by multiplex PCR using primers to amplify codon 315 region of the katG gene and MTb specific IS6110 region. The two detection probes complementary to the 5' end of each amplified fragment was conjugated with gold nanoparticles (20 nm) and coupled with the above amplified PCR products were applied on the sample pad. The hybridization of the amplified target regions to the respective capture probes takes place when the sample moves towards the absorption pad. Positive hybridization is indicated by red colour lines. RESULTS: The three immobilized capture probes on the strip (for the detection of TB, katG wild type and mutation) were 100 and 96.6% specific and 100 and 92.1% sensitive respectively. CONCLUSION: The AuNP based lateral flow assay was capable of differentiating the specific mutation and the wild type along with MTb identification within 3 h.


Assuntos
Doenças Transmissíveis/diagnóstico , Nanotecnologia/métodos , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Catalase/genética , Doenças Transmissíveis/tratamento farmacológico , DNA Bacteriano/isolamento & purificação , DNA Bacteriano/metabolismo , Ouro/química , Humanos , Isoniazida/uso terapêutico , Limite de Detecção , Nanopartículas Metálicas/química , Reação em Cadeia da Polimerase Multiplex , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Sri Lanka , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico
17.
Environ Pollut ; 253: 358-364, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31325880

RESUMO

Spread of pathogens in pig farms not only causes transfection of diseases to other pigs or even farmers working in the farms, but also induces pollution to the living atmospheric environment of the residents around the farm. Therefore, it is necessary to establish a rapid and simple monitoring method. In this study, full genome sequences of common viruses were analyzed in pig farms, in combination with the design of primers, optimization of the reaction parameters, so as to establish a multiplex RT-PCR assay for the identification of classical swine fever virus (CSFV), Japanese encephalitis virus (JEV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus Type 2 (PCV-2), porcine pseudorabies virus (PRV) and porcine parvovirus virus (PPV), which are common in pig farms. This method has a minimal detectable concentration of 10-3 ng/µL, which is highly specific. Furthermore, multiplex RT-PCR was applied to examine air samples from 4 pig farms located in different cities of China. The results were in line with those obtained by single PCR. Therefore, this study can be expected to provide essential technique support for the early warning mechanism as well as disease prevention and control system against the major viruses.


Assuntos
Microbiologia do Ar , Poluentes Atmosféricos/análise , Monitoramento Ambiental , Vírus , Animais , China , Circovirus , Primers do DNA , Fazendas , Reação em Cadeia da Polimerase Multiplex , Vírus da Síndrome Respiratória e Reprodutiva Suína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/virologia
18.
Medicine (Baltimore) ; 98(25): e16109, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31232956

RESUMO

RATIONALE: It is rare to find 22q11.2 deletion syndrome with pseudohypoparathyroidism in children. Furthermore, the phenotypic spectrum of this disorder varies widely. PATIENT CONCERNS: A patient was diagnosed with pseudohypoparathyroidism at age 14 years because of convulsions, hypocalcemia, hyperphosphatemia, normal parathyroid hormone levels, and basal ganglia calcifications. Thereafter, the child presented with symptoms of nephrotic syndrome; subsequently, he was diagnosed with nephrotic syndrome at the local hospital. DIAGNOSIS: At our hospital, multiplex ligation-dependent probe amplification confirmed that the patient had 22q11.2 deletion syndrome. INTERVENTIONS: The patient continued to be treated with calcium supplements. OUTCOMES: Seizure activity and proteinuria ceased. LESSONS: Signs of this syndrome include delayed speech development due to velofacial dysfunction, recurrent croup attacks during early childhood due to latent hypocalcemia, and mild dysmorphic features. The findings of this patient indicated that 22q11.2 deletion syndrome may include a wide spectrum of clinical findings and that this diagnosis needs to be considered for all patients presenting with hypocalcemia, regardless of age.


Assuntos
Síndrome de DiGeorge/diagnóstico , Pseudo-Hipoparatireoidismo/diagnóstico , Adolescente , Síndrome de DiGeorge/genética , Humanos , Hiperfosfatemia/etiologia , Hipocalcemia/etiologia , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Pseudo-Hipoparatireoidismo/complicações , Pseudo-Hipoparatireoidismo/genética , Convulsões/etiologia
19.
Parasit Vectors ; 12(1): 313, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234884

RESUMO

BACKGROUND: Alveolar echinococcosis is a dangerous zoonotic disease caused by larval forms of Echinococcus multilocularis. In its life-cycle, the principal definitive host is the red fox; however, domesticated carnivorous animals (dogs and cats) can also act as definitive hosts. Until now, there were no data concerning this infection in cats in Poland. The aim of this study was to estimate the prevalence of E. multilocularis in cats and dogs originating from rural areas and animal shelters in a region characterised by a high prevalence of E. multilocularis in red foxes. METHODS: Samples of faeces were collected from 67 cats and 268 dogs from a rural area (villages and animal shelters) of a highly endemic region in southeastern Poland. Samples were examined using nested PCR (E. multilocularis), multiplex PCR (E. multilocularis, Taenia spp.) and PCR [E. granulosus (s.l.)]. Additionally, faeces were examined microscopically (flotation). Moreover, intestines from 110 red foxes shot in the investigated area were examined (sedimentation and counting technique). RESULTS: Positive PCR results for E. multilocularis were obtained in 4 cats (6.0%) and 4 dogs (1.5%). There were no significant differences between groups of animals (from a shelter and with an owner) concerning the prevalence of E. multilocularis in both cats and dogs. Taenia spp. were found in 10 cats (14.9%) (Taenia taeniaeformis and T. hydatigena) and 26 dogs (9.7%) (T. hydatigena, T. serialis, T. taeniaeformis, T. crassiceps, T. pisiformis and T. ovis) and Mesocestoides litteratus was found in 4 cats (6.0%) and 3 dogs (1.1%). All samples were negative for E. granulosus by PCR. Taking into consideration PCR and flotation results, 29 cats (43.3%) and 73 dogs (27.2%) were infected with helminths (26.9 and 11.9%, respectively, were infected with tapeworms). The highly endemic status of the investigated area was confirmed by examination of red foxes: 48.2% of examined red foxes were infected with E. multilocularis. CONCLUSIONS: To the best of our knowledge, this study reports the presence of E. multilocularis in cats for the first time in Poland and confirms the role of dogs in this infection in highly endemic areas.


Assuntos
Doenças do Gato/epidemiologia , Gatos/parasitologia , Doenças do Cão/epidemiologia , Equinococose/veterinária , Doenças Endêmicas/veterinária , Animais , Doenças do Gato/parasitologia , Doenças do Cão/parasitologia , Cães/parasitologia , Equinococose/epidemiologia , Echinococcus granulosus/isolamento & purificação , Echinococcus multilocularis/isolamento & purificação , Fezes/parasitologia , Feminino , Raposas/parasitologia , Masculino , Reação em Cadeia da Polimerase Multiplex , Polônia/epidemiologia , Prevalência , População Rural , Taenia/isolamento & purificação , Teníase/epidemiologia , Teníase/veterinária
20.
J Med Microbiol ; 68(8): 1211-1218, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31225788

RESUMO

INTRODUCTION: Lower respiratory tract infections (LRTIs), particularly those acquired in hospitals, are an important cause of childhood morbidity and mortality. Understanding the aetiology and epidemiology of LRTIs is necessary for clinical management, reduction of antibiotic usage, vaccine development and prevention of nosocomial infection. AIM: In this study, we aimed to detect 13 viruses and atypical bacteria in nasopharyngeal secretion specimens from hospitalized children with LRTIs. METHODOLOGY: From January 2014 to December 2016, nasopharyngeal secretion specimens were prospectively collected from 3232 children aged between 1 and 72 months. Nucleic acid was extracted and analysed using the SureX13 respiratory pathogen multiplex kit as per the manufacturer's instructions. RESULTS: A total of 2874 (88.9 %) children tested positive for viral and/or atypical bacterial infections, and 965 (29.9 %) were co-infected with multiple pathogens. The most frequently detected respiratory tract pathogens (RTPs) were rhinovirus, respiratory syncytial virus, parainfluenza virus and adenoviruses. The rates of RTP and co-infection positivity in the toddler group were significantly higher than those in the infant and preschool groups. CONCLUSION: The SureX13 respiratory pathogen multiplex kit has the ability to effectively detect a range of RTPs in hospitalized paediatric patients with LRTIs.


Assuntos
Bactérias/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/normas , Kit de Reagentes para Diagnóstico/normas , Infecções Respiratórias/diagnóstico , Vírus/isolamento & purificação , Fatores Etários , Bactérias/classificação , Bactérias/genética , Criança Hospitalizada/estatística & dados numéricos , Pré-Escolar , China/epidemiologia , Coinfecção/diagnóstico , Coinfecção/epidemiologia , Coinfecção/microbiologia , Coinfecção/virologia , Feminino , Humanos , Lactente , Masculino , Nasofaringe/microbiologia , Nasofaringe/virologia , Prevalência , Infecções Respiratórias/epidemiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Análise de Sequência de DNA , Vírus/classificação , Vírus/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA